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Tanner and Tanner: Citrate treatment in PKD rats 1861
perchloric acid. The samples were centrifuged, and the
supernatants were then filtered through a 10,000 molecular
weight cutoff UltraFuge filter (Micron Separations,
Westboro, MA, USA).
Histology
The remaining kidney was fixed by retrograde aortic
perfusion with a solution of 3% paraformaldehyde, 137
mmol/L NaCl, 2.7 mmol/L KCl, 1.5 mmol/L KH 2 PO 4 , 4
mmol/L Na 2 HPO 4 , and 2 mmol/L picric acid . at a perfusion
pressure of 150 to 170 mm Hg. The kidney was
kept in the same fixative solution for several days in the
refrigerator and then weighed, sliced with a razor blade,
immersed in 0.1 mol/L cacodylate solution (pH 7.25),
and embedded in paraffin. Sections were stained with
hematoxylin and eosin. The degree of cystic disease (size
of cysts, interstitial widening and fibrosis, presence of
inflammatory cells) was evaluated blindly using an arbitrary
scale of 0 to 4, where 0 represents the normal
condition and 4 represents severe changes.
Chemical analyses
Polyfructosan (a synthetic inulin) in plasma and urine
was determined by an anthrone method [12]. PAH was
determined by Bratton and Marshall’s method [13]. Ci-
trate in plasma, urine, and tissue samples was determined
spectrophotometrically using a citrate lyase method
(Boehringer Mannheim, Indianapolis, IN, USA). Recovery
of citrate added to plasma averaged 99 6% (N
11). When kidney tissue was analyzed for citrate, we
observed a 100 5% (N 25) recovery of an internal
citrate standard at five minutes after adding the citrate
lyase, indicating completion of the reaction. The absor-
bance readings, however, continued to drift appreciably
with time, probably because of the presence of nicotin-
amide adenine dinucleotide (NADH) oxidase activity in
kidney cortex extracts. To correct for this “creep,” we
measured absorbance every 5 minutes for a total of 20
minutes after adding the citrate lyase and extrapolated
the absorbance back to time zero [14]. Ammonia was
determined using a glutamate dehydrogenase-based kit
(Boehringer Mannheim). Recovery of ammonia added
to plasma averaged 100 1.4% (N 4). Phosphate in
plasma was determined by the Fiske-SubbaRow method.
Potassium in urine and plasma and calcium in plasma,
urine, and kidney tissue were determined using an atomic
absorption spectrophotometer (model 951; Instrumenta-
tion Laboratory, Wilmington, MA, USA). Tissue sam-
ples were homogenized in a solution containing 4% (vol/
vol) butanol, 0.2 mol/L HCl, and 36 mmol/L LaCl 3 [15]
using a Polytron homogenizer (Brinkmann Instruments,
Westbury, NY, USA). Recovery of calcium added to
tissue samples averaged 103 3% (N 9). Samples
and standards were always prepared in the same matrix
solutions.
Calculations
The GFR was calculated from the rate of excretion
of polyfructosan divided by the plasma polyfructosan
concentration. Renal plasma flow (RPF) was calculated
from the PAH clearance divided by the PAH extraction
ratio ((arterial minus renal venous plasma [PAH])
arterial plasma [PAH]). Renal blood flow (RBF) was
calculated from this formula: RBF RPF/(1 hemato-
crit). Renal citrate consumption was calculated from
RPF (arterial plasma [citrate] renal vein plasma
[citrate]) minus urinary citrate excretion rate. Tubular
reabsorption was calculated from the filtered load minus
the excretion rate. Peritubular uptake of citrate was cal-
culated from the renal citrate consumption minus the
rate of tubular citrate reabsorption. Fractional excretion
was calculated from the excretion rate divided by the
filtered load. Renal ammonia production was calculated
from the RBF (renal venous blood [ammonia] arte-
rial blood [ammonia]) plus the urinary excretion rate.
The partial pressure of cortical tissue ammonia (P NH3 )
was estimated from the renal vein total NH 4 concentration
and arterial blood pH [16, 17] and this formula:
P NH3 (total NH 4 22.09)/(10 pK pH )
where the pK for NH 4 is 9.02. The pH of renal venous
and arterial blood are assumed to be the same, and the
solubility coefficient () for ammonia is 0.91 [18].
Statistical methods
Data presented are means SD. They were analyzed
by two-way analysis of variance (ANOVA), after a pre-
liminary test for homogeneity of variances. Individual
groups were compared with the Bonferroni method. If
variances were heterogeneous, the Kruskal–Wallis test
and Dunn’s test were used to compare means. A P value
of less than 0.05 was considered significant.
RESULTS
Overall function in six-month-old rats
The effects of KCitr consumption in six-month-old
normal rats and rats with PKD are summarized in Table
1. The most remarkable finding is that GFR was com-
pletely normal in rats with PKD that had been treated
with KCitr, whereas GFR was only 37% of normal in
rats with PKD that had consumed tap water. RBF also
was normal in the rats with PKD that had been treated
with KCitr and was about half of normal in untreated
rats with PKD. KCitr treatment had no effect on body
weight or blood pressure, in agreement with our earlier
findings at three months of age [7].
A comparison of water-drinking normal rats and wa-
ter-drinking rats with PKD (Table 1) shows that the
animals with cystic disease had heavier kidneys, higher